Clostridium difficile infection (CDI) is one of the most common healthcare-associated infections (HAI). Despite rising CDI incidence in children, studies of pediatric CDI are relatively limited. Diagnosing CDI in children is very challenging, particularly because children are commonly asymptomatic carriers of C. difficile. Nucleic acid amplification tests (NAATs) are most commonly used to diagnose CDI at US children's hospitals, primarily because of adult studies suggesting suboptimal sensitivity of toxin enzyme immunoassays (EIAs). NAATs are highly sensitive and also detect C. difficile in children who are asymptomatic carriers. Thus, NAAT positivity does not necessarily indicate that C. difficile is the cause of a child's diarrhea, and CDI misdiagnosis among carriers is a common consequence of using NAATs. To overcome the limitations of both NAATs and EIAs, a recently developed ultrasensitive toxin assay is under investigation in adult patients. CDI misdiagnosis leads to unnecessary antibiotic utilization for CDI among carriers, increasing healthcare costs, antibiotic resistance, and adverse events. CDI misdiagnosis also biases investigation of pediatric CDI. Therefore, improved CDI diagnostic strategies are necessary to improve patient care for this common HAI and reduce misclassification bias in future CDI investigation in children. To address these challenges, the proposed study aims to comprehensively evaluate various laboratory methodologies to better differentiate CDI and carriage, resulting in improved CDI diagnosis in children. In addition, using whole genome sequencing (WGS), we aim to identify genomic differences between strains causing CDI and carriage that may reveal genomic targets unique to strains causing carriage or CDI that can be adapted for CDI diagnostic testing. Specifically, we aim to: (1) Define the optimal testing strategy for diagnosing CDI in children wit diarrhea; (2) Determine the relative propensity of various NAATs to identify C. difficile carriage in children without diarrhea; and (3) Using WGS, (a) Identify C. difficile genotypes isolated from children; and (b) Identify genomic biomarkers of CDI and carriage. With co-mentorship from Alan Hauser, MD, PhD and Dale Gerding, MD, Larry Kociolek, MD seeks to build upon his previous CDI translational research experience. Dr. Kociolek will learn genomic methods of C. difficile characterization and methods for applying pathogen genomic data to patient-oriented research. Dr. Kociolek's overarching career goal is to become a leader in the investigation of pediatric CDI and coordinate collaborative efforts to reduce CDI burden in children. The excellent integrated research infrastructure at the Ann & Robert H. Lurie Children's Hospital of Chicago and Northwestern University Feinberg School of Medicine will facilitate achievement of his academic goals. Future research directions include coordinating a multi-center pediatric study to validate optimal CDI diagnostic strategies and subsequently identifying both host and pathogen factors that contribute to CDI and its complications in children. These data are essential for guiding future clinical trials of preventive and therapeutic strategies for CDI in children.